Sphingosine derivatives and medicinal composition

ABSTRACT

The present invention intends to provide a derivative of sphingosine analogue that is able to regulate the functions of sphigolipid, and its pharmaceutical compositions. 
     The present invention is the derivatives of sphingosine analogues represented by the general formula (I) described below.                    
     In the formula, R 1  and R 2 , which are the same or different each other, are hydrogen, alkyl groups having 1-4 carbon atoms, or acyl groups having 2-5 carbon atoms. R 3  and R 4 , which are the same or different each other, are hydrogen or hydroxyl groups; or R 3  and R 4  make up a covalent bond. X 1  is —(CH 2 ) n —CO—NH—CH(R 5 )—R 6  or —(CH 2 ) m —O—CO—CH(R 7 )—R 8 .

This application is a 371 of PCT/JP98/04095 filed Sep. 11, 1998.

TECHNICAL FIELD

The present invention relates to derivatives of sphingosine analoguesand their pharmaceutical compositions, which are useful as drugs for thetreatment of fungal infections, allergic diseases, immune disorders,etc.

PRIOR ART

Sphingosine is a compound having the chemical structure shown in thegeneral formula described below, in which Y¹ is hydrogen. It is knownthat various sphingolipids having sphingosine as a constituent arewidely distributed in the living body including on the surface of cellmembranes of cells in the nervous system. Also we knowglycosphingolipids, which have one or several kinds of sugars as Y¹ viaa glycoside bond linked to a ceramide having a fatty acid bound to theamino group of sphingosine via a peptide bond, and sphingophospholipids,including sphingomyelin, which have a phosphoric acid and a base such ascholine or ethanolamine as Y¹ linked to the above-mentioned ceramide.

A sphingolipid is one of the lipids having important roles in the livingbody. We know a disease called lipidosis which is caused by accumulationof a specified sphingolipid in the body concomitant with theabnormalities in the metabolic pathways due to defect of an enzyme andothers. Attractive effects of sphingolipids present on the cellmembranes include functions in the regulation of cell growth anddiscrimination of each cell; functions in the developments anddifferentiation; functions in nerves; involvement in the infections andmalignancy of cells; and others. Lots of physiological roles of sucheffects remain to be solved. Recently a possibility that ceramide, aderivative of sphingosine, has an important role in the mechanism ofcell signal transduction was indicated, and studies about its effectsand so forth on apoptosis and cell cycle have been actively performed.

Fungi and plants have sphingolipids and the major sphingosine containedin these organisms has the formula described below. It is known thatthese lipids have important roles in the cell growth of fungi andplants, but details of the roles remain to be solved.

Recently it has been known that derivatives of sphingolipids and theirrelated compounds exhibit a variety of biological activities throughinhibition or stimulation of the metabolism pathways. These compoundsinclude inhibitors of protein kinase C, inducers of apoptosis,immuno-suppressive compounds, antifungal compounds, and the like.Substances having these biological activities are expected to be usefulcompounds for various diseases.

SUMMARY OF THE INVENTION

In view of the above present states, the present invention intends toprovide a derivative of sphingosine analogue that is able to regulatethe functions of sphigolipid, and its pharmaceutical compositions.

In the course of a search for novel biologically active compounds, theinventors discovered novel biologically active compounds TKR1785's,which showed antifungal activity and immunosuppressive activity, in theculture broth of TKR1785 strain belonging to Penicillium sp.

In this specification, TKR1785's are compounds shown in the followinggeneral formula (II). TKR1785's described above include TKR1785-I shownas the following formula (IIa) and TKR1785-II (IIb).

The inventors succeeded in preparation of novel sphingosine analoguesrepresented by the following formula (III) by hydrolysis of TKR1785'sdescribed above.

Furthermore they succeeded in synthesis of peptide derivatives describedin the following general formula (Ia) using, as the starting material,the compound described by the above formula (III) or the compounddescribed by the following formula (IIIa) that was prepared from thecompound described by the above formula (III) used as the startingmaterial. They found that these compounds showed biological activitiessuch as antifungal activity and immunosuppressive activity.

In the formula, R³ and R⁴, which are the same or different each other,are hydroxyl groups or hydrogen; or R³ and R⁴ make up a covalent bond.Y² is —COOH or —CH₂OH.

In the formula, R¹ is hydrogen, an alkyl group having 1-4 carbon atoms,or an acyl group having 2-5 carbon atoms. R³ and R⁴, which are the sameor different each other, are hydrogen or hydroxyl groups; or R³ and R⁴make up a covalent bond. X² is —CO—NH—CH(R⁵)—R⁶ or —CH₂—O—CO—CH(R⁷)—R⁸.R⁵ is hydrogen, a hydroxyl group, or an alkyl group having 1-4 carbonatoms which may have a hydroxyl group. R⁶ is —CH₂OH, —COOH, —CONH₂, or—CO—NH—CH(R⁹)—R¹⁰. R⁹ is hydrogen, a hydroxyl group, or an alkyl grouphaving 1-4 carbon atoms which may have a hydroxyl group. R¹⁰ is —CH₂OH,—COOH, —CONH₂, or —CO—NH—CH(R¹¹)—R¹². R¹¹ is hydrogen, a hydroxyl group,or an alkyl group having 1-4 carbon atoms which may have a hydroxylgroup. R¹² is —CH₂OH, —COOH, or —CONH₂. R⁷ is hydrogen, a hydroxylgroup, or an alkyl group having 1-4 carbon atoms which may have ahydroxyl group. R⁸ is —CH₂OH, —NH₂, or —NH—CO—CH(R¹³)—R¹⁴.

R¹³ is hydrogen, a hydroxyl group, or an alkyl group having 1-4 carbonatoms which may have a hydroxyl group. R¹⁴ is —CH₂OH, —NH₂, or—NH—CO—CH(R¹⁵)—R¹⁶. R¹⁵ is hydrogen, a hydroxyl group, or an alkyl grouphaving 1-4 carbon atoms which may have a hydroxyl group. R¹⁶ is —NH₂ or—CH₂OH.

However, the compound which has hydrogen as R¹ and R³, a hydroxyl groupas R⁴, and —CH₂—CO—NH—CH(R⁵)—R^(6 as X) ², in which R⁵ is —CH(CH₃)₂ or—CH—(CH₃)C₂H₅, and R⁶ is —CO—NH—CH(R⁹)—R¹⁰ in which R⁹ and R¹⁰ are—CH₂OH, is excluded.

Also, the inventors succeeded in preparation of peptide derivativesdescribed by the following general formula (Ib) from various sphingosineanalogues which have the chemical structures similar to the aboveformula (III), including above sphingosine and phytosphingosine, andfound that these showed biological activities similar to those of thecompounds described by the above general formula (Ia), resulting incompletion of the present invention.

In the formula, R¹, R³, and R⁴ are the same as described above. X³ isthe same as X² of the above formula (Ia).

DETAILED DESCRIPTION OF THE INVENTION

Following is the present invention now described in detail.

Derivatives of sphingosine analogues of the present invention arerepresented by the general formula (I) described below.

In the formula, R¹ and R², which are the same or different each other,are hydrogen, alkyl groups having 1-4 carbon atoms, or acyl groupshaving 2-5 carbon atoms. R³ and R⁴, which are the same or different eachother, are hydrogen or hydroxyl groups; or R³ and R⁴ make up a covalentbond. X¹ is —(CH₂)_(n)—CO—NH—CH(R⁵)—R⁶ or —(CH₂)_(m)—O—CO—CH(R⁷)—R⁸. Then means an integral number ranging from 0 to 3. R⁵ is hydrogen, ahydroxyl group, or an alkyl group having 1-4 carbon atoms which may havea hydroxyl group. R⁶ is —CH₂OH, —COOH, —CONH₂, or —CO—NH—CH(R⁹)R¹⁰. R⁹is hydrogen, a hydroxyl group, or an alkyl group having 1-4 carbon atomswhich may have a hydroxyl group. R¹⁰ is —CH₂OH, —COOH, —CONH₂, or—CO—NH—CH(R¹¹)—R¹². R¹¹ is hydrogen, a hydroxyl group, or an alkyl grouphaving 1-4 carbon atoms which may have a hydroxyl group. R¹² is —CH₂OH,—COOH, or —CONH₂. The m means an integral number ranging from 1 to 3. R⁷is hydrogen, a hydroxyl group, or an alkyl group having 1-4 carbon atomswhich may have a hydroxyl group. R⁸ is —CH₂OH, —NH₂, or—NH—CO—CH(R¹³)—R¹⁴. R¹³ is hydrogen, a hydroxyl group, or an alkyl grouphaving 1-4 carbon atoms which may have a hydroxyl group. R¹⁴ is —CH₂OH,—NH₂, or —NH—CO—CH(R¹⁵)—R¹⁶. R¹⁵ is hydrogen, a hydroxyl group, or analkyl group having 1-4 carbon atoms which may have a hydroxyl group. R¹⁶is —NH₂ or —CH₂OH.

However, the compound which has hydrogen as R¹, R², and R³, a hydroxylgroup as R⁴, and —CH₂CO—NH—CH(R⁵)—R⁶ as X¹, in which R⁵ is —CH(CH₃)₂ or—CH(CH₃)C₂H₅, and R⁶ is —CO—NH—CH(R⁹)—R¹⁰ in which R⁹ and R¹⁰ are—CH₂OH, is excluded.

The alkyl group having 1-4 carbon atoms is not limited, and examples aremethyl, ethyl, propyl, i-propyl, n-butyl, and t-butyl group.

The acyl group having 2-5 carbon atoms is not limited, and examples areacetyl, propionyl, n-butyryl, and valeryl group.

The derivatives of sphingosine analogues of the present invention arecompounds represented by the general formula (I) described above, whichincludes compounds shown by the above general formula (Ia) or the abovegeneral formula (Ib), and the examples are the compounds described inTable 1 shown below.

TKR1785's represented by the above general formula (II), TKR1785-I andTKR1785-II, are prepared by culturing a strain enabling to produceTKR1785's and belonging to Penicillium sp., and isolating them from thecultured broth thereafter. The strain useful to produce TKR1785'sdescribed above is exemplified by Penicillium sp. TKR1785 (referred toas “strain TKR1785” thereafter). That is, strain TKR1785 is inoculatedinto a nutrient medium and cultured in the liquid to obtain TKR1785'sdescribed above.

The present inventors deposited the above strain TKR1785 under depositnumber of FERM BP-5788 (original date of deposit: May 17, 1995; date ofrequest for transfer to international deposit: Jan. 17, 1997) atNational Institute of Bioscience and Human Technology (address; 1-3Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan (Zip-code 305)).

The culture described above is carried out at 15 to 25° C. preferably,and the incubation for 3 to 11 days usually gives a sufficientproduction. TKR 1785's accumulated in the cultured product are obtainedby purification utilizing their physicochemical and biologicalproperties. When the above purification can be carried out by a methodusing high performance liquid chromatography, a silica gelchemically-bonded by, for example, octadecyl, octyl, or phenyl group, apolystyrene type porus-polymer gel, or the like is available. The mobilephase includes an aqueous solution of water-soluble organic solvent, forexample, aqueous methanol, aqueous acetonitrile, or the like.

The compound represented by the formula (III) to be used for preparationof the compounds represented by the above general formula (I) of thepresent invention can be prepared from hydrolysis of TKR1785'srepresented by the above general formula (II). For example, the compoundcan be obtained by acid hydrolysis of TKR1785-I of the above formula(IIa), e.g. decomposition under the condition of 6N HCl at 110° C. forovernight, which is used for hydrolysis of the peptide bond, followed byneutralizing the reaction solution and being adjusted to alkali. Thecompound prepared can be isolated by neutralization of the reactionsolution again, extraction with an organic solvent such as chloroform, amixture of chloroform/methanol and the like, and if necessary, furtherpurification using an adsorption chromatography using silica gel or areversed phase partition chromatography using a chemical-bonded silicagel.

The carboxyl group of the compound represented by the above formula(III) can be converted to an amide by methylation of the carboxyl groupfollowed by ammonolysis, or to a hydroxy group by reduction usinglithium aluminum hydride (LiAlH₄), sodium borohydride (NaBH₄), or thelike, resulting in the compound having —CH₂OH as Y²in the above generalformula (IIIa).

The lactone represented by the below formula (IV) can be obtained whenTKR1785-1 is subjected to acid hydrolysis under the condition similar tothat described above, followed by concentration, and then purification.

The compound, in which R³ and R⁴ of the compound shown by the abovegeneral formula (IIIa) together make a covalent bond, resulting in adouble bond between carbon number 4 and carbon number 5, can be easilyobtained from the lactone of the above general formula (IV) bydehydration by treating with concentrated sulfuric acid or by reactingwith thionyl chloride in pyridine and followed by alkaline hydrolysis.

The compound, in which R³ and R⁴ of the compound shown by the abovegeneral formula (IIIa) together make a covalent bond and its Y² is COOH,can be prepared by the alkaline hydrolysis of the above compound.

Sphingosine analogues that are able to be used to prepare thederivatives represented by the above general formula (I) of the presentinvention include not only the compounds shown by the above generalformula (IIIa) but also commercially available phytosphingosine,sphingosine, a compound derived from these compounds by hydrogenation ofthe double bond thereof (sphinganine), a compound derived from thesecompounds by hydration of the double bond thereof, and a compoundderived from these compounds by modification of the terminal hydroxylgroup to a caroboxyl group.

To prepare the compounds represented by the above general formula (I),lots of protecting groups used widely for the peptide synthesis can beproperly utilized. Such protecting groups, for example, includeprotecting groups of the amino group, protecting groups of the carboxylgroup, and protecting groups of the hydroxyl group.

The above protecting group of the amino group is not particularlyrestricted, and includes, for example, t-butoxycarbonyl (Boc) group,trichloroethoxycarbonyl (Troc) group, and the like.

The above protecting group of the carboxyl group is not particularlyrestricted, and includes, for example, phenacyl (Pac) group, benzyl(Bzl) group, and the like.

The above protecting group of the hydroxyl group is not particularlyrestricted, and includes, for example, Bzl group, acetyl group, methylgroup, trimethylsilyl group, and the like.

The above protecting group is respectively removed in need by a knownelimination reaction corresponding to each group or its appliedelimination reaction, resulting in modification to an objectivecompound. In the case that protecting groups that can be eliminated by adifferent condition are used, a selective modification can be easilycarried out by performing the above elimination reaction.

For example, in order to prepare a derivative, a compound represented bythe general formula (I), using a sphingosine analogue having a carboxylgroup at the terminal, the carboxyl group of the sphingosine analogue islinked to the amino group of an amino acid or an amino alcohol by apeptide bond. Methods to make the above peptide bond include, forexample, a method which comprises activating the carboxyl group bywater-soluble carbodiimide (WSCD) and HOBt, a method which comprisesactivating the carboxyl group by making carboxyazide bydiphenylphosphorylazide, and a method using(benzotriazolyloxy)tris-(dimethylamino)phosphonium fluorophosphate(BOP).

Also, in order to prepare a derivative, a compound represented by thegeneral formula (I), using a sphingosine analogue having a hydroxylgroup at the terminal, the hydroxyl group of the sphingosine analogue islinked to the carboxyl group of an amino acid or a carboxylic acid by anester bond. Methods to make the above ester bond include, for example, amethod using N,N′-dicyclohexylcarbodiimide (DCC) in the presence of acatalyst like dimethyl aminopyridine (DMAP) or 4-pyrrolidinopyridine,and the mixed acid anhydride method using 2,4,6-trichlorobenzoylchloride.

Many kinds of sphingosine analogues including sphingosine andphytosphingosine can be used as the starting material to prepare thederivative represented by the above general formula (I) of the presentinvention, and an acylated or alkylated product of the sphingosineanalogues can also be utilized, because the derivative represented bythe above general formula (I) includes a product with the amino groupthereof acylated or alkylated.

The above acylation can be carried out by a conventional method usingacid anhydride or acid chloride and so forth, and the above alkylationcan be performed by a method using sodium hydride and alkyl iodide.

The derivative of sphingosine analogues of the present invention showsthe biological activity as shown in Table 2 described below and usefulas a pharmaceutical composition.

The derivative of sphingosine analogues of the present invention caneach be put to use as such or in the form of a pharmacologicallyacceptable salt in medicinal applications. There is no particularlimitation on the type of pharmacologically acceptable salt. Thus, thesalt includes salts of mineral acids such as hydrochloric acid, sulfuricacid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid,etc., salts of organic acids such as formic acid, acetic acid, tartaricacid, lactic acid, citric acid, fumaric acid, maleic acid, succinicacid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,toluenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid,etc., and salts of alkali metals or alkaline earth metals, such assodium, potassium, calcium, etc.

To administer the derivative of sphingosine analogues of the presentinvention or its pharmacologically acceptable salt, as a drug, thederivative of sphingosine analogues of the present invention can beadministered either as such or in the form of a pharmaceuticalcomposition containing typically 0.1 to 99.5%, preferably 0.5 to 90%thereof in a pharmaceutically acceptable, non toxic and inert carrier toanimals inclusive of humans.

The carrier mentioned above includes solid, semisolid or liquiddiluents, fillers, other formulation auxiliaries, etc. and such carrierscan be used alone or in combination.

The above-mentioned pharmaceutical composition is preferablyadministered in unit dosage forms and can be administered orally,parenterally, topically (e.g. transdermally) or rectally. Of course,those pharmaceutical compositions should be administered in dosage formssuited for the respective route of administration.

For administration of the derivative of sphingosine analogues or itspharmacologically acceptable salt of the present invention, as a drug,the dose as an antifungal agent or an antiallergic agent is preferablyselected with reference to patient factors such as age and body weight,route of administration, nature and severity of disease, etc. Usually inman, however, the daily dose of the active ingredient for an adultpatient is 10 to 2000 mg. While a daily dose lower than the above rangemay be sufficient in some cases, a dose higher than the range may berequired in other cases. When a high dose is used, the daily dosage ispreferably administered in several divided doses.

The oral administration mentioned above can be made using solid,powdery, or liquid dosage forms such as bulc powders, powders, tablets,dragees, capsules, drops, subligual tablets, etc.

For the above-mentioned parenteral administration, liquid unit dosageforms for subcutaneous, intramuscular, or intravenous administration,typically solutions and suspensions, can be employed. These preparationscan be manufactured by suspending or dissolving a predetermined amountof the derivative of sphingosine analogue or its pharmacologicallyacceptable salt of the present invention, in a nontoxic liquid carriersuitable for injection, such as an aqueous medium or an oily medium, andsterilizing the resulting suspension or solution.

The above-mentioned topical administration (e.g. transdermaladministration) can be carried out using a variety of topical dosageforms such as liquids, creams, powders, pastes, gels, and ointments.These dosage forms can be manufactured by using a predetermined amountof the derivative of sphingosine analogues of the present invention or apharmacologically acceptable salt thereof, in combination with one ormore of the perfume, coloring agent, filler, surfactant, humectant,emollient, gelatinizer, carrier, preservative, stabilizer, etc.,suitable for topical dosage formulations.

The rectal administration can be made using, for example, suppositorieseach mixing a predetermined amount of the derivative of sphingosineanalogues or its pharmacologically acceptable salt of the presentinvention, with a low-melting solid base such as higher esters, e.g.myristyl palmitate, polyethylene glycol, cacao butter, or a mixture ofthem.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples illustrate the present invention in furtherdetail but are not intended to define the scope of the invention.

EXAMPLE 1 Synthesis of Compound (1) from Sphingosine (see Scheme 1)

1) Preparation of N-Boc derivative: Sphingosine (10.0 mg, 33.4 μmol) wasdissolved in dioxane-water (10:1, 300 μl), Boc—ON (12.7 mg, 50.2 μmol)and N,N-dhisopropylethylamine (DIEA) (8.8 μl, 50.2 μmol) were addedthereto at room temperature, and the mixture was stirred for 5 hours.After addition of ethyl acetate, the reaction mixture was washed with10% aqueous solution of citric acid and then with saturated aqueoussolution of NaCl. The ethyl acetate layer was dried over magnesiumsulfate, concentrated under reduced pressure, and purified by silica gelthin layer chromatography (TLC) (chloroform-methanol 19: 1), yieldingcolorless powder of compound (1a) (7.7 mg).

FAB-MS: m/z 400 (M+H)

¹H-NMR (500 MHz, DMSO-d₆) δ6.18 (d), 5.51 (m), 5.40 (m), 4.76 (d), 4.39(t), 3.83 (m), 3.49-3.38 (m), 1.93 (m), 1.36-1.20 (m), 0.84 (t), 0.00(s).

2) Preparation of a TBDMS derivative of the primary alcohol: Compound(1a) (5.7 mg, 14.3 μmol) was dissolved in dichloromethane (CH₂Cl₂) (400μl), dimethylamiinopyridine (DMAP) (1 mg), triethylamine (NEt₃) (2.4 μl,17.1 μmol), and t-butyldimethylsilyl chloride (TBDMS-Cl) (2.4 mg, 15.7μmol) were added thereto, and the mixture was stirred at roomtemperature for 16 hours. The reaction mixture was purified by TLC(chloroform-methanol 100: 1), yielding colorless oil of compound (1b)(6.7 mg).

FAB-MS: m/z 514 (M+H)

¹H-NMR (500 MHz, DMSO-d₆) δ6.18 (d), 5.50 (m), 5.41 (m), 4.74 (d), 3.82(m), 3.67 (m), 3.52-3.39 (m), 1.94 (m), 1.35-1.19 (m), 0.84 (m), 0.00(s).

3) Preparation of an N-Boc derivative of the secondary alcohol: Compound(1b) (2.0 mg, 3.89 μmol) was dissolved in CH₂Cl₂ (400 μl), DMAP (1 mg)and Boc₂O (1.0 mg, 4.67 μmol) were added thereto, and the mixture wasstirred for 5.5 hours. After additional addition of Boc₂O (2.0 mg, 9.34μmol), the mixture was further stirred for 17.5 hours. The reactionmixture was purified by TLC (chloroform-methanol 200: 1), yieldingcolorless oil of compound (1c) (2.4 mg).

FAB-MS: m/z 614 (M+H)

¹H-NMR (500 MHz, DMSO-d₆) δ6.65 (d), 5.65 (m), 5.41 (m), 4.92 (t), 3.73(m), 3.52 (m), 1.97 (m), 1.41-1.22 (m), 0.84 (m).

4) Selective removal of TBDMS: Compound (1c) (2.4 mg, 3.91 μmol) intetrahydrofuran (THF)-acetic acid-water (1:2:1, 1.5 ml) was stirred atroom temperature for 17.5 hours. After concentration under reducedpressure, the reaction mixture was purified by TLC (chloroform-methanol50: 1), yielding colorless oil of compound (1d) (2.0 mg).

FAB-MS: m/z 500 (M+H)

¹H-NMR (500 MHz, DMSO-d₆) δ6.58 (d), 5.64 (m), 5.39 (m), 4.91 (t), 4.62(t), 3.66 (m), 3.44 (m), 1.96 (m), 1.40-1.22 (m), 0.84 (t).

5) Coupling of sphingosine and valine: Compound (Id) (3.7 mg, 7.40μmol), Z—Val—OH (2.8 mg, 11.1 μmol; Z: benzyloxycarbonyl group), andDMAP (0.45 mg, 3.70 μmol) were dissolved in CH₂Cl₂ (300 μl), DCC (2.3mg, 11.1 μmol) was added thereto under ice-cooling, and the mixture wasstirred for 10 minute and then at room temperature for further 16 hours.After filtration, the reaction mixture was concentrated under reducedpressure, and purified by TLC (chloroform-methanol 100:1), yieldingcolorless oil of compound (1e) (4.0 mg).

FAB-MS: m/z 733 (M+H)

¹H-NMR (500 MHz, DMSO-d₆) δ7.66 (d), 7.36-7.28 (m), 6.97 (d), 5.67 (m),5.55 (d), 5.39 (m), 5.03 (d), 4.88 (t), 3.93 (m), 1.96 (m), 1.71 (m),1.60 (m), 1.50 (m), 1.38-1.00 (m), 0.84 (m).

6) Synthesis of compound (1f): To a solution of compound (1e) (4.0 mg,5.46 μmol) in ethyl acetate (20 ml), palladium-black (20 mg) was added,and the mixture was bubbled with hydrogen gas at room temperature for 90minutes. After filtering off the catalyst, the mixture was concentratedunder reduced pressure, resulting in elimination of the Z group andreduction of the double bond, and the compound (1f) (3.8 mg) wasobtained.

FAB-MS: m/z 890 (M+H)

¹H-NMR (500 MHz, DMSO-d₆) δ8.40 (d), 6.99 (d), 5.07 (m), 4.61 (m),4.33-3.85 (m), 2.07 (m), 1.54-1.22 (m), 0.84 (m).

7) Synthesis of O—Boc-glyceric acid: To a solution of glyceric acid(92.4 mg, 0.871 mmol) in acetone (300 μl), NEt₃ (14.6 μl, 1.05 mmol) andphenacyl bromide (PacBr) (203 mg, 1.05 mmol) was added underice-cooling, and the mixture was stirred for 1 hour and then at roomtemperature for further 4 hours. After concentration under reducedpressure, the residue was dissolved in ethyl acetate, and washed withwater, saturated aqueous solution of sodium hydrogen carbonate and thensaturated aqueous solution of NaCl. The ethyl acetate layer was driedover magnesium sulfate, concentrated, and then dried under reducedpressure. The residue was dissolved in ethyl acetate (2 ml), DMAP (36.4mg, 0.298 mmol) and Boc₂O (260.3 mg, 1.193 mmol) were added theretounder ice-cooling, and the mixture was stirred for 5 minutes and forfurther 4 hours at room temperature. Ethyl acetate was added to thereaction mixture, which was then washed with 10% aqueous solution ofcitric acid, saturated aqueous solution of NaCl, saturated aqueoussolution of sodium hydrogen carbonate, and then saturated aqueoussolution of NaCl. The ethyl acetate layer was dried over magnesiumsulfate, concentrated under reduced pressure, and purified by TLC(benzene-ethyl acetate 30: 1), yielding the Pac-ester of —Boc-glycericacid (47.6 mg). This compound (42.8 mg, 0.101 mmol) was dissolved in 90%aqueous solution of acetic acid solution (5 ml), Zn powder (330.2 mg,5.05 mmol) was added thereto under ice-cooling, and the mixture wasstirred for 15 minutes and then 1 hour at room temperature. Afterfiltering off insoluble materials, the reaction mixture was concentratedunder reduced pressure, and the resulting residue was dissolved in ethylacetate, washed with 10% aqueous solution of citric acid, and thensaturated aqueous solution of NaCl. The ethyl acetate layer was driedover magnesium sulfate, concentrated under reduced pressure, andpurified by TLC (chloroform-methanol-acetic acid 95:5:3), yieldingcolorless powder of O—Boc-glyceric acid (19.7 mg).

FAB-MS: m/z 305 (M−H)

¹H-NMR (500 MHz, DMSO-d₆) δ5.05 (m), 4.34 (m), 1.41 (d).

8) Coupling of compound (1f) and glyceric acid: Compound (1f) (3.1 mg,5.18 μmol) and O—Boc-glyceric acid (2.4 mg, 7.76 μmol) were dissolved indimethyl formamide (DMF) (500 μl), HOBt (0.84 mg, 6.21 μmol) and WSCD(1.0 μl, 5.69 μmol) were added thereto under ice-cooling, and themixture was stirred for 3 hours. Ethyl acetate was added to the reactionmixture, which was then washed with 10% aqueous solution of citric acid,saturated aqueous solution of NaCl, saturated aqueous solution of sodiumhydrogen carbonate, and then saturated aqueous solution of NaCl. Theethyl acetate layer was dried over magnesium sulfate, concentrated underreduced pressure, the residue obtained was purified by TLC(chloroform-methanol 200:1), and the protected compound (1) wasobtained. TFA (500 μl ) was added to the product obtained, and themixture was concentrated under reduced pressure after stirring for 2hours at room temperature, yielded colorless oil of compound (1) (0.5mg) shown in Table 1.

FAB-MS: m/z 489 (M+H)

EXAMPLE 2 Synthesis of compound (2)

Compound (1d) (9.3 mg, 18.6 μmol), Fmoc—Val—OH (9.5 mg, 27.9 μmol;9-fluorenylmethoxycarbonyl group), and DMAP (1.1 mg, 9.31 μmol) weredissolved in CH₂Cl₂(500 μl ), DCC (5.8 mg, 27.9 μmol) was added theretounder ice-cooling followed by stirring for 45 minutes and then at roomtemperature for further 19 hours. After filtration and concentrationunder reduced pressure, the reaction mixture was purified by TLC(chloroform-methanol 200:1), yielding a compound (10.7 mg). To thecompound obtained (8.6 mg, 10.5 μmol), 30% piperidine/DMF solution (1ml) was added under ice-cooling followed by stirring for 30 minutes andthen at room temperature for further 1 hour to remove the Fmocprotecting group selectively. The reaction mixture was neutralized by 1NHCl under ice-cooling, concentrated under reduced pressure, and purifiedby TLC (chloroform-methanol 19:1), yielding the coupling product (4.5mg) between the protected-sphingosine (1d) and valine (Fmoc—Val—OH).

FAB-MS: m/z 599 (M+H)

The product obtained (5.4 mg, 9.02 μmol) and O—Boc-glyceric acid (4.1mg, 13.5 μmol) were dissolved in DMF (500 μl), HOBt (1.5 mg, 10.8 μmol)and WSCD (1.8 μl, 9.92 μmol) were added thereto under ice-coolingfollowed by stirring for 3 hours. Ethyl acetate was added to thereaction mixture, which was then washed with 10% aqueous solution ofcitric acid, saturated aqueous solution of NaCl, saturated aqueoussolution of sodium hydrogen carbonate, and then saturated aqueoussolution of NaCl. The ethyl acetate layer was dried over magnesiumsulfate, concentrated under reduced pressure, and purified by TLC(chloroform-methanol 100:1), yielding the protected compound (2) (4.3mg). To the product obtained, TFA (500 μl) was added, and the mixturewas stirred for 2 hours at room temperature, concentrated under reducedpressure, yielding colorless oil of compound (2) (1.8 mg) shown in Table1.

FAB-MS: m/z 487 (M+H)

EXAMPLE 3 Synthesis of compound (3)

Phytosphingosine was used instead of sphingosine used in Example 1 asthe starting material, and gave colorless oil (1.5 mg) of compound (3)shown in Table 1 by steps similar to those for synthesis of compound (1)in Example 1.

FAB-MS: m/z 505 (M+H)

Reference Example 1 Synthesis of TKR1785-I

1) Preparation of isopropylidene derivative of compound (III). Compound(III) (5.0 mg, 14.5 μmol) was suspended in acetone (900 μl), and acetonedimethylacetal (150 μl) and dl-camphor sulfonic acid (1 mg) were addedthereto followed by stirring for 1 hour at room temperature. Thereaction mixture was neutralized with NEt₃ (10 μl) and concentratedunder reduced pressure. The residue was purified by TLC [the lower layerof a mixture of chloroform-methanol-water (8:3:1)], and gave theobjective compound (amount 2.8 mg, yield 51%) as colorless oil.

FAB-MS: m/z 386 (M+H)

2) Preparation of the trichloroethoxycarbonyl (Troc) derivative: Thecompound obtained in 1) (1.4 mg, 3.6 μmol) was dissolved in pyridine(100 μl), and trichloroethoxycarbonyl chloride (1.5 μl, 10.9 μmol) wasadded thereto under ice-cooling followed by stirring for 30 min. underice-cooling and then for 1 hour at room temperature. The reactionmixture was purified by TLC (the lower layer of a mixture ofchloroform-methanol-water, 8:3:1), and gave the objective compound(amount 1.0 mg, yield 42%) as colorless powder.

FAB-MS: m/z 734 (M−H)

3) Synthesis of TKR1785-I. The compound obtained in 2) described above,Troc derivative (5.0 mg, 6.0 μmol) andHCl.H₂N—CH(CH(CH₃)₂)—CONH—CH(CH₂OBzl)—CH₂OBzl (4.8 mg, 12.0 μmol), whichwas separately prepared using Boc—NH—CH(CH₂OH)CH₂OBzl as the startingmaterial, were dissolved in CH₂Cl₂ (100 μl), and appropriate amount ofWSCD and HOBt were added thereto as described in Examples 1-8). Themixture was stirred for 30 min. under ice-cooling and stirred overnightat room temperature. The reaction mixture was purified by TLC(chloroform-methanol 19:1), and gave the objective compound (amount 3.9mg, yield 60%) as colorless powder.

FAB-MS: m/z 1088 (M+H)

The product (3.9 mg, 3.6 μmol) was dissolved in methanol (3 ml), andpalladium-black (15 mg) was added thereto followed by stirring at roomtemperature for 90 minutes with bubbling of hydrogen gas. Afterfiltering off the catalyst from the reaction mixture, the filtrate wasconcentrated under reduced pressure, yielding colorless powder.

FAB-MS: m/z 908 (M+H)

Then, the product obtained was dissolved in 90% acetic acid aqueoussolution (2 ml), and Zn powder (100 mg) was added thereto underice-cooling followed by stirring for 10 minutes under ice-cooling andfor 2 hours at room temperature. After filtering off insolublematerials, the filtrate was concentrated under reduced pressure,yielding TKR1785-I (amount 1.1 mg) as colorless powder.

FAB-MS: m/z 518 (M+H)

EXAMPLE 4 Synthesis of Compound (4)

1) Oxidation of Compound (1d): Pyridinium dichromate (12.8 mg, 34 μmol)suspended in DMF (50 μl) was added to a solution of compound (1d) (4.8mg, 9.6 μmol) in DMF (50 μl) followed by stirring for 24 hours at roomtemperature. After addition of water, the mixture was stirred for 10minutes, extracted with chloroform, and the extract was concentratedunder reduced pressure. The residue was purified by TLC, yieldingcolorless solid (amount 2.5 mg, yield 51%).

FAB-MS: m/z 514 (M+H)

2) Synthesis of Compound (4): The compound (2.0 mg, 3.9 lμmol) obtainedin the above 1), and HCl.H₂N—CH(CH(CH₃)₂)—CONH—CH(CH₂OTroc)—CH₂OTroc(4.5 mg, 7.8 μmol), which was separately prepared using—Boc—NH—CH(CH₂OH)CH₂OH as the starting material, were coupled similarlyto Examples 1-8). Purification of the reaction mixture by TLC gave theprotecting derivative of compound (4) (amount 2.8 mg, yield 70%) ascolorless powder.

FAB-MS: m/z 1037 (M+H)

This compound obtained (2.8 mg, 2.7 μmol) was dissolved in 90% aceticacid aqueous solution (2 ml), and zinc powder (100 mg) was added theretounder ice-cooling followed by stirring for 10 minutes under ice-coolingand for further 1 hour at room temperature. After filtering offinsoluble materials, the filtrate was concentrated under reducedpressure, yielding colorless powder.

FAB-MS: m/z 687 (M+H)

Thereafter, to the compound obtained, trifluoroacetic acid (TFA) (500μl) was added under ice-cooling. The mixture was left for 1 hour underice-cooling, and concentrated under reduced pressure, yielding thecompound (4) (amount 1.0 mg) as colorless powder.

FAB-MS: m/z 487 (M+H)

EXAMPLE 5 Synthesis of Compound (5)

Compound (1d) (4.0 mg, 8.00 μmol), O—Boc-glyceric acid (3.0 mg, 12.0μmol), and DMAP (0.49 mg, 4.00 μmol) were dissolved in CH₂Cl₂ (300 μl),and DCC (2.5 mg, 12.0 μmol) wad added thereto under ice-cooling followedby stirring for 10 minutes and then for additional 14 hours at roomtemperature. After filtration, the reaction mixture was concentratedunder reduced pressure and purified by TLC (chloroform-methanol, 200:1).TFA (500 μl) was added to the compound obtained, and after stirring for2 hours at room temperature the mixture was concentrated under reducedpressure, yielding compound (5) (1.4 mg) shown in Table 1 as colorlessoil.

FAB-MS: m/z 388 (M+H)

EXAMPLE 6

To the coupling compound (4.4 mg, 7.3 μmol) of the protected-sphingosine(1d) obtained in Example 2 and valine, TFA (500 μl) was added followedby stirring for 3 hours at room temperature. The reaction mixture wasconcentrated under reduced pressure, yielding the compound (6) (2.3 mg)shown in Table 1 as colorless oil.

FAB-MS: m/z 399 (M+H)

EXAMPLE 7 Synthesis of Compound (7)

The carboxyl group of compound (III) was reduced by LiAlH₄, yielding aprimary OH, and followed by changing to a N-Boc derivative. Then theprimary OH was protected by a t-butyldimethylsilyl (TBDMS) group, thesecondary OH was by O—Boc group, and then the TBDMS of the primary OHwas removed, yieldingH₃C—(CH₂)₁₂—CH(OBoc)—CH₂—CH(OBoc)—CH(NBBoc)—CH₂—CH₂OH. This compound(3.5 mg, 5.5 μmol) and Fmoc—Ile—OH (2.5 mg, 7.1 μmol) were used as thestarting materials to be coupled in the similar way to Example 2, andFmoc and Boc were removed from the product, yielding compound (7) (1.7mg) shown in Table 1 as colorless oil.

FAB-MS 445 (M+H)

EXAMPLE 8 Synthesis of Compound (8)

Boc—Ser(Bzl)-ol (300 mg, 1.07 mmol) was dissolved in DMF (2 ml), and NaH(50% oil suspension; 77.3 mg, 1.61 mmol) was added under ice-coolingthereto followed by stirring for 30 minutes. After addition of benzylbromide (165 μl, 1.39 mmol), the mixture was stirred for 10 minutesunder ice-cooling, and stirred for 1 hour at room temperature. Aqueoussolution of 10% citric acid was added to the reaction mixture, and ethylacetate added for extraction. The ethyl acetate layer was washed withsaturated aqueous solution of sodium hydrogen carbonate, then saturatedaqueous solution of NaCl, dried over magnesium sulfate, and concentratedunder reduced pressure, yielding the dibenzyl derivative of Boc—Ser-ol.The Boc group of this compound was removed and sequentially coupled toBoc—Val—OH and then to Boc—Ala—OH. The Boc group of the product wasremoved. The product (9.5 mg, 16.9 μmol) obtained was coupled to thecompound (5.7 mg, 11.1 μmol) obtained in the step 1) of the synthesis ofcompound (4), and the protecting groups were removed by an ordinarymethod, yielding compound (8) (0.5 mg) shown in Table 1 as colorlessoil.

FAB-MS: m/z 559 (M+H)

EXAMPLE 9 Synthesis of Compound (9)

Compound (1) (2.0 mg, 4.1 μmol) was dissolved in methanol (200 μl), andacetic anhydride (60 μl) was added thereto under ice-cooling. Themixture was stirred for 18 hours at room temperature. The reactionmixture was concentrated under reduced pressure and the residue waspurified by TLC (developed and eluted with a solution ofchloroform-methanol, 19:1), yielding compound (9) (1.5 mg) shown inTable 1 as colorless oil.

FAB-MS: m/z 531 (M+H)

TABLE 1 Compounds R¹ R² R³ R⁴ X¹ (1) H H H H

(2) H H — (double — bond)

(3) H H OH H

(4) H H — (double — bond)

(5) H H — (double — bond)

(6) H H — (double — bond)

(7) H H H OH

(8) H H H H

(9) —CO— CH₃ H H H

Test Example Biological Characteristics

Antifungal activity and immunosuppressive activity of the productderivatives of sphingosine analogues were investigated by the followingmethod. The results are set forth in Table 2.

Measurement of Antifungal Activity

The test microorganism used was Cryptococcus neoformans TIMM 0354. YNBGliquid medium (Difco yeast nitrogen base 0.67%, glucose 1%) was used andthe minimum growth inhibitory concentration (SIC) was determined afterculturing for 2 days at 30° C. in the presence of the serial two-folddilution of the compound. The minimum growth sub-inhibitoryconcentration (μg/ml) was determined as the minimum concentrationcausing partial growth inhibition of the microorganism and also shown inTable 2 in parentheses.

Measurement of Immunosuppressive Activity

We measured inhibitory activity of mixed lymphocytes reaction (MLR) anddetermined the 50% inhibitory activity. Lymphocytes used are T cellsfrom spleen cells of C57BL/6 mice and BALB/c mice.

TABLE 2 Antifungal activity Immunosuppressive activity MIC (μg/ml) IC50(μg/ml) Compound (1) 50 (25) 3.25 Compound (2)   25 (12.5) 1.86 Compound(5) 50 (25) 5 Compound (6) 50 (25) 1.55 Compound (8) (50) 0.44

TABLE 3 Scheme 1

INDUSTRIAL APPLICABILITY

The derivatives of sphingosine analogues of the present inventionconsist of the composition described above, and have antifungal activityand immunomodulatory activity including immunosuppression. Thus they canbe used as antifungal agents and immunoregulatory agents.

What is claimed is:
 1. Derivatives of sphingosine analogues representedby the general formula (I) described below.

In the formula, R¹ and R², which are the same or different each other,are hydrogen, alkyl groups having 1-4 carbon atoms, or acyl groupshaving 2-5 carbon atoms. R³ and R⁴, which are the same or different eachother, are hydrogen or hydroxyl groups; or R³ and R⁴ make up a covalentbond. X¹ is —(CH₂)_(n)—CO—NH—CH(R⁵)—R⁶ or —(CH₂)_(m)—O—CO—CH(R⁷)—R⁸. Then means an integral number ranging from 0 to
 3. R⁵ is hydrogen, ahydroxyl group, or an alkyl group having 1-4 carbon atoms which may havea hydroxyl group. R⁶ is —CH₂OH, —COOH, —CONH₂, or —CO—NH—CH(R⁹)—R¹⁰. R⁹is hydrogen, a hydroxyl group, or an alkyl group having 1-4 carbon atomswhich may have a hydroxyl group. R¹⁰ is —CH₂OH, —COOH, —CONH₂, or—CO—NH—CH(R¹¹)—R¹². R¹¹ is hydrogen, a hydroxyl group, or an alkyl grouphaving 1-4 carbon atoms which may have a hydroxyl group. R¹² is —CH₂OH,—COOH, or —CONH₂. The m means an integral number ranging from 1 to
 3. R⁷is hydrogen, a hydroxyl group, or an alkyl group having 1-4 carbon atomswhich may have a hydroxyl group. R⁸ is —CH₂OH, —NH₂, or—NH—CO—CH(R¹³)—R¹⁴. R¹³ is hydrogen, a hydroxyl group, or an alkyl grouphaving 1-4 carbon atoms which may have a hydroxyl group. R¹⁴ is —CH₂O,—NH₂, or —NH—CO—CH(R¹⁵)R¹⁶. R¹⁵ is hydrogen, a hydroxyl group, or analkyl group having 1-4 carbon atoms which may have a hydroxyl group. R¹⁶is —NH₂ or —CH₂OH. The compound which has hydrogen as R¹, R², and R³, ahydroxyl group as R⁴, and —CH₂—CO—NH—CH(R⁵)—R⁶ as X¹, in which R⁵ is—CH(CH₃)₂ or —CH—(CH₃)C₂H₅, and R⁶ is —CO—NH—CH(R⁹)—R¹⁰ in which R⁹ andR¹⁰ are —CH₂OH, is excluded.
 2. Pharmaceutical compositions comprisingthe derivative of sphingosine analogue as claimed in claim 1 or itspharmacologically acceptable salt as an effective ingredient.